scholarly journals Opening the side exit pores of ClpP by lowering the pH of proteolytic chamber coupled with substrate hydrolysis

2021 ◽  
Author(s):  
Leehyeon Kim ◽  
Byung-Gil Lee ◽  
Min Kyung Kim ◽  
Do Hoon Kwon ◽  
Hyunmin Kim ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Activation Mechanism ◽  
Product Release ◽  
Physiological Processes ◽  
Cryo Electron Microscopy ◽  
Structural Details ◽  
The Relationship ◽  
Small Chemical ◽  
Substrate Hydrolysis

The ClpP serine peptidase is a tetradecameric degradation machine involved in many physiological processes. It becomes a competent ATP-dependent protease with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause dysregulation and activation of ClpP without ATPases, and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed the structural details, conformational changes, and activation mechanism. Although product release by the side exit pores has been proposed, the detailed driving force for product release remains elusive. Here, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures revealed various conformational states at different pH conditions. To understand the conformational change for product release, we investigated the relationship between substrate hydrolysis and the pH lowering process. Our data, together with previous findings, provide insight into the molecular mechanism of product release by ClpP self-compartmentalizing protease.

scholarly journals Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1

2020 ◽  
Author(s):  
Yanxin Liu ◽  
Ming Sun ◽  
Alexander G. Myasnikov ◽  
Daniel Elnatan ◽  
Nicolas Delaeter ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Activation Mechanism ◽  
Client Protein ◽  
Protein Maturation ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Client Proteins ◽  
Rate Limiting

AbstractHsp90 is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of cellular “client” proteins. The ATP-driven client maturation process is regulated by a large number of co-chaperones. Among them, Aha1 is the most potent activator of Hsp90 ATPase activity and thus dramatically affects Hsp90’s client proteins. To understand the Aha1 activation mechanism, we determined full-length Hsp90:Aha1 structures in six different states by cryo-electron microscopy, including nucleotide-free semi-closed, nucleotide-bound pre-hydrolysis, and semi-hydrolyzed states. Our structures demonstrate that the two Aha1 domains can each interact with Hsp90 in two different modes, uncovering a complex multistep activation mechanism. The results show that Aha1 accelerates the chemical step of ATP hydrolysis like a conventional enzyme, but most unusually, catalyzes the rate-limiting large-scale conformational changes of Hsp90 fundamentally required for ATP hydrolysis. Our work provides a structural framework to guide small molecule development targeting this critical modulator of client protein maturation.

scholarly journals Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Valeria Kalienkova ◽  
Vanessa Clerico Mosina ◽  
Laura Bryner ◽  
Gert T Oostergetel ◽  
Raimund Dutzler ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Energy Barrier ◽  
Bound State ◽  
Activation Mechanism ◽  
Regulation Mechanism ◽  
Closed Cavity ◽  
X Ray ◽  
Functional Assays ◽  
Cryo Electron Microscopy ◽  
Lipid Nanodiscs

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+ binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement.

scholarly journals Structural and Functional Diversity among Agonist-Bound States of the GLP-1 Receptor

2021 ◽  
Author(s):  
Brian P. Cary ◽  
Peishen Zhao ◽  
Tin T. Truong ◽  
Sarah J. Piper ◽  
Matthew J. Belousoff ◽  
...  
Keyword(s):  
Bound States ◽  
Conformational Dynamics ◽  
Structural Data ◽  
Signal Propagation ◽  
Cryo Electron Microscopy ◽  
Conformational Plasticity ◽  
Glucagon Like Peptide 1 ◽  
Peptide Agonist ◽  
The Relationship ◽  
G Protein Coupled

ABSTRACTRecent advances in G protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multi-dimensional signal propagation mediated by these receptors is, in part, dependent on their conformational mobility. However, the relationship between receptor function and static structures determined via crystallography or cryo-electron microscopy is not always clear. This study examines the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide-1 receptor (GLP-1R), an important clinical target. We employ variants of the peptides GLP-1 and exendin-4 to explore the interplay between helical propensity near the agonist N-terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an exendin-4 analogue, the GLP-1R and Gs protein revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.

scholarly journals Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM

2018 ◽  
Author(s):  
Valeria Kalienkova ◽  
Vanessa Clerico Mosina ◽  
Laura Bryner ◽  
Gert T. Oostergetel ◽  
Raimund Dutzler ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Bound State ◽  
Activation Mechanism ◽  
Regulation Mechanism ◽  
Closed Cavity ◽  
X Ray ◽  
Functional Assays ◽  
Cryo Electron Microscopy ◽  
Lipid Environment ◽  
Lipid Nanodiscs

AbstractScramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+-binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement.Impact statementcryo-EM reveals the properties of distinct conformations occupied during activation of the lipid scramblase nhTMEM16 and provides new insights into its interactions with the lipid environment.

scholarly journals Structural basis for activation and allosteric modulation of full-length calcium-sensing receptor

2021 ◽  
Vol 7 (23) ◽  
pp. eabg1483
Author(s):  
Tianlei Wen ◽  
Ziyu Wang ◽  
Xiaozhe Chen ◽  
Yue Ren ◽  
Xuhang Lu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Hormone Secretion ◽  
Allosteric Modulation ◽  
Full Length ◽  
Structural Basis ◽  
Endocrine Disorders ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Class C

Calcium-sensing receptor (CaSR) is a class C G protein–coupled receptor (GPCR) that plays an important role in calcium homeostasis and parathyroid hormone secretion. Here, we present multiple cryo–electron microscopy structures of full-length CaSR in distinct ligand-bound states. Ligands (Ca2+ and l-tryptophan) bind to the extracellular domain of CaSR and induce large-scale conformational changes, leading to the closure of two heptahelical transmembrane domains (7TMDs) for activation. The positive modulator (evocalcet) and the negative allosteric modulator (NPS-2143) occupy the similar binding pocket in 7TMD. The binding of NPS-2143 causes a considerable rearrangement of two 7TMDs, forming an inactivated TM6/TM6 interface. Moreover, a total of 305 disease-causing missense mutations of CaSR have been mapped to the structure in the active state, creating hotspot maps of five clinical endocrine disorders. Our results provide a structural framework for understanding the activation, allosteric modulation mechanism, and disease therapy for class C GPCRs.

scholarly journals Cryo-EM structure of mycobacterial cytochrome bd reveals two oxygen access channels

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weiwei Wang ◽  
Yan Gao ◽  
Yanting Tang ◽  
Xiaoting Zhou ◽  
Yuezheng Lai ◽  
...  
Keyword(s):  
Rational Design ◽  
Pathogenic Bacteria ◽  
Structural Topology ◽  
Antimicrobial Drugs ◽  
E Coli ◽  
Cytochrome Bd ◽  
Cryo Electron Microscopy ◽  
Functional Mechanisms ◽  
The Relationship ◽  
Potential Oxygen

AbstractCytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

Behavior as a window on physiology: a simple apparatus for recording caterpillar feeding.

1992 ◽  
Vol 262 (6) ◽  
pp. S9 ◽  
Author(s):  
E Bowdan
Keyword(s):  
Feeding Behavior ◽  
Control Mechanisms ◽  
Inexpensive Method ◽  
Simple Apparatus ◽  
Physiological Processes ◽  
Wide Range ◽  
Relationship Of ◽  
The Relationship

Regulation of feeding is a fundamental element of homeostasis. This is reflected in the similarity of control mechanisms in a wide range of animals, including insects and humans. A close examination of feeding behavior can illuminate the physiological processes driving regulation. A simple, inexpensive method for recording fine details of feeding by caterpillars is described. Possible experiments, interpretation of the data, and the relationship of observations to the underlying physiology, are outlined.

scholarly journals The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy

2015 ◽  
Vol 89 (23) ◽  
pp. 12108-12117 ◽  
Author(s):  
Jian Guan ◽  
Stephanie M. Bywaters ◽  
Sarah A. Brendle ◽  
Hyunwook Lee ◽  
Robert E. Ashley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Human Papillomavirus ◽  
Conformational Changes ◽  
Vaccine Development ◽  
Contact Point ◽  
Human Papillomavirus 16 ◽  
Cryo Electron Microscopy ◽  
C Terminus ◽  
L1 Protein

ABSTRACTThe human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers.IMPORTANCEHuman papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

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